EPO is a glycoprotein hormone which stimulates the proliferation, differentiation and maturation of erythroid precursor cells to mature red blood cells. EPO has been purified (Miyake et al., (1977) J. Biol. Chem. 252: 5558) and molecularly cloned (Lin et al. (1985) Proc. Natl. Acad. Sci. USA 82: 7580), and recombinant hEPO has been used successfully in the treatment of anemia due to end stage renal disease. Clinical use of EPO has also been reported in the treatment of anemia associated with AIDS, rheumatoid arthritis, hematological malignancies, and prematurity, and to increase the yield of autologous blood collected preoperatively.
Recombinant hEPO has a molecular mass of 30.4 kD, of which 40% is carbohydrate. Studies of the nature and function of the glycosylation of EPO have determined that the majority of the oligosaccharide chains of hEPO are fucose-containing, sialylated tetraantennary oligosaccharides. The glycosylation structure of human urinary EPO and recombinant EPO produced in Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells and human B-lymphoblastic cells is similar but not identical.
Glycosylation appears to play a role in solubility, biosynthesis and secretion of EPO, and in vivo metabolism of EPO. The in vivo metabolic role of glycosylation, and in particular the presence of sialic acid, has been investigated. It has been demonstrated that urinary and recombinant desialylated EPO lose in vivo activity due to rapid hepatic clearance. From these and similar studies it has been determined that sialic acid caps the penultimate galactose residue and thus protects EPO from clearance by hepatic asialoglycoprotein (galactosyl) receptors. Accordingly, loss of terminal sialic acid residues from the oligosaccharides of EPO exposes the galactose residues, resulting in rapid clearance of desialylated (also referred to as asialylated) EPO from the plasma via binding to the hepatic receptor.
Although desialylated EPO is essentially biologically inactive in vivo due to its rapid clearance, its bioactivity is maintained in established in vitro assays. Standard assays for determining bioactivity of EPO in vitro include measurement of incorporation of tritiated thymidine by splenic erythroblasts from phenylhydrazine-treated anemic mice (Krystal (1983) Exp. Hematol. 20: 649) or cells of a human pluripotent leukemia cell line (Lewis et al. (1989) Exp. Hematol. 17: 102), measurement of incorporation of .sup.59 Fe into cultured bone marrow cells (Goldwasser et al. (1975) Endocrinology 97: 315), and measurement of the growth of EPO-dependent cell lines (Kitamura et al. (1989) Blood 73: 375).
Because the in vitro assays cannot discriminate between sialylated and desialylated EPO, such assays are not useful in quantitating the population of EPO which would be expected to be active in vivo. For example, if an EPO-containing sample also contains a significant amount of desialylated EPO, the in vitro assays would provide an overestimate of the in vivo activity. Accordingly, in vivo assays are used to measure the in vivo activity of EPO. Specifically, EPO activity is measured by incorporation of .sup.59 Fe into erythroblasts of polycythemic mice (Cotes et al. (1961) Nature 191: 1065) or starved rats (Goldwasser et al. (1975) Methods Enzymol. 37: 109).
In a currently used assay which is a modification of the procedure of Cotes et al., female mice are exposed to hypobaric pressure for fourteen days. Endogenous red cell formation is suppressed by the polycythemia produced through exposure to reduced pressure. As the polycythemic state persists after hypoxia, any new red blood cell formation is attributable to the administration of exogenous EPO. The test samples and EPO standards are then injected subcutaneously into the conditioned mice. Forty-eight hours after EPO injection, .sup.59 Fe is administered. Blood samples are drawn after another forty-eight hours, and radioactivity is quantitated. The quantity of radioactivity is directly proportional to the injected dose of standard EPO; the in vivo activity of unknown samples is calculated from a standard curve.
The in vivo bioassay is widely recognized as the only true measure of in vivo biological activity, since it measures both the circulating life and proliferative activity of EPO. However, the in vivo assay suffers from significant disadvantages in that it is labor-intensive, expensive, time-consuming, and subject to animal-to-animal variation.
The present invention provides an in vitro method of quantitating the in vivo activity of EPO which overcomes the disadvantages of the prior art methods.